Normal wound healing involves the formation of scars and fibrous tissues. Their structures consist largely of collagen fibrils. Although collagen is required in wound repair, collagen often accumulates in excessive amounts and impairs the normal function of the affected tissue. Such excessive amounts of collagen become an important event in scarring of the skin following burns or other traumatic injury, as well as in fibrosis of the liver, lungs and other organs following injury. Because of the central role of collagen in the pathogenesis of fibrosis, there has been considerable interest in agents capable of inhibiting collagen accumulation in fibrotic diseases. Potential target sites for inhibiting collagen accumulation include transcription of the genes, translation of the mRNAs, and some of the post-translational enzymes involved in the biosynthesis of collagen [G. C. Fuller, 1981, J. Med. Chem., 24: 651-658; M. Trojanowska et al., 1998, J. Mol. Med., 76: 266-274]. Here we will focus our attention to circumvent collagen accumulation in fibrosis by inhibiting cross-link levels. To understand the rationale behind attempts to circumvent collagen accumulation by inhibiting cross-link levels it is necessary to get an insight into the way cross-linking of collagen molecules occurs.
Biosynthesis of collagen is a multistep process, resulting in extensive modification of the molecule (FIG. 1). The different modifications of the molecule are catalyzed by various enzymes with an intra- or extracellular localization [K. Kadler, 1994, Protein Profile, 1: 515-638]. One of the steps in the biosynthesis of collagen is hydroxylation of certain proline residues in the triple helix by prolyl 4-hydroxylase (EC 1.14.11.2) and prolyl 3-hydroxylase (EC 1.14.11.7) as well as hydroxylation of certain lysine residues in the triple helix and telopeptides by lysyl hydroxylase (EC 1.14.11.4). In a next step, hydroxylysine residues in the triple helix can be subjected to glycosylation by glycosyl transferases. Hydroxylation of proline and lysine are essential for a proper functioning of collagen. There is hardly any variation in the level of prolyl hydroxylation within a specific collagen type. In fact, the hydroxylation level of prolyl residues of each fibrillar collagen type is close to a maximum; underhydroxylation of proline residues results in a non-functional molecule with a weakened triple helix that can easily be degraded. In contrast, there are large variations in lysyl hydroxylation within the same collagen type (e.g. type I collagen) between the different tissues. The functional significance of this variation is unknown, but under- and overhydroxylation of lysine residues is associated with several connective tissue disorders, such as Ehlers-Danlos type VI syndrome [A. Ihme et al., 1984, J. Invest. Dermatol., 83: 161-165] and osteogenesis imperfecta [W. G. Cole, 1994, Bone Miner. Res., 8: 167-2041].
Once the collagen molecule is secreted, the propeptides are cleaved off by procollagen N-peptidase (EC 3.4.24.14) and procollagen C-peptidase (EC 3.4.24.19), resulting in a mature molecule consisting of a triple helix with a short telopeptide at both termini. The mature molecules aggregate spontaneously into microfibrils. Further stabilization of the molecules occurs by means of cross-links. Cross-linking is initiated by conversion of specific lysine or hydroxylysine residues of the telopeptides into the aldehydes allysine and hydroxyallysine, respectively, by the enzyme lysyl oxidase (EC 1.4.3.13) [H. M. Kagan, 1994, Path. Res. Pract., 190: 910-919]. The aldehydes subsequently react with lysine or hydroxylysine residues of the triple helix to give characteristic difunctional cross-links. These cross-links eventually mature into tri- or tetra-functional cross-links [D. R. Eyre, 1987, Meth. Enzymol., 144: 115-139; A. J. Bailey et al., 1998, Mech. Ageing Developm., 106: 1-56]. Two related routes for the formation of cross-links have been described, one based on allysine from the telopeptides, the other based on the hydroxyallysine of the telopeptides. Each route results in chemically distinct cross-links. For the nomenclature and origin of some of the most common cross-links we refer to FIG. 2.
Hydroxylation of lysine in the triple helix of collagen occurs exclusively on lysine present in the helical amino acid sequence Gly-X-Lys-Gly; a lysine in the X position is not hydroxylated [K. Kadler, 1994, Protein Profile, 1: 515-638]. The hydroxylated lysine in the telopeptides is embedded in an entirely different amino acid sequence; in view thereof, the existence of another enzyme, whose substrate would be the non-helical telopeptide region, has occasionally been proposed [J. E. Gerriets et al., 1996, Biochim. Biophys. Acta, 1316: 121-131; J. E. Gerriets et al., 1993, J. Biol. Chem., 286: 25553-25560; L. Knott et al, 1997, Biochem. J., 322: 535-542; M. J. Barnes et al., 1974, Biochem. J., 139: 461-468; P. M. Royce and M. J. Barnes, 1985, Biochem. J., 230: 475-480]. However, the circumstantial evidence that an enzyme exists that specifically hydroxylates the telopeptide lysine has mostly been ignored and/or questioned. We will show later on that hydroxylation of telopeptide lysine residues is indeed under separate control from that of helical lysine residues, i.e. hydroxylation of telopeptide lysine and helical lysine is independently and specifically controlled.
Essentially all stages of collagen biosynthesis have been used as targets for the pharmacological control of collagen accumulation in fibrosis, the most important ones being the inhibition of prolyl hydroxylation (e.g. by incorporation of prolyl analogues into the triple helix instead of prolyl) [H. M. Hanauske-Abel, 1991, J. Hepatol., 13 Suppl. 3: S8-S16] and cross-linking. Here we will focus our attention to the inhibition of cross-link formation as a tool to decrease collagen accumulation in fibrotic tissues. Collagen molecules that are not cross-linked are more easily degraded by proteinases [C. A. Vater et al., 1979, Biochem. J., 181: 639-645], thus facilitating the removal of collagen. Two routes have been described to achieve lower cross-link levels: compounds that are able to inhibit lysyl oxidase (so-called lathyrogens, an example being xcex2-aminopropionitrile) and compounds that essentially block aldehydes (such as penicillamine, cysteine, and other analogues) [M. E. Nimni, 1983, Sem. Arthr. Rheum., 13: 1-86]. Both routes result in a decrease of both allysine and hydroxyallysine derived cross-links. The consequence of inhibition of lysyl oxidase is that aldehydes cannot be formed in the telopeptides; as a result, cross-link formation does not take place (FIG. 1-2). Blocking the aldehydes also effectively inhibits cross-link formation, as blocked aldehydes cannot participate in the formation of intramolecular and intermolecular cross-links. Although lathyrogens and compounds that react with aldehydes are potent antifibrotic agents, concentrations needed to display antifibrotic effects have toxic effects, thus limiting its clinical use as antifibrotic drugs. An additional drawback is, that unphysiological collagen is formed, showing inferior biomechanical properties due to the lower cross-link levels. This unphysiological collagen is not only formed by proliferating fibroblasts but also by the cells surrounding the injury, thus impairing the biomechanical quality of healthy tissue as well. In this invention we will describe a method that does not show this drawback: agents can be developed that selectively inhibit the formation of unwanted hydroxyallysine cross-links in collagen of the fibrotic lesions, leaving synthesis of allysine cross-links in the intact tissue and healing wound unaffected.
In yet another approach minoxidil, an agent capable of suppressing fibroblast proliferation and expression of lysyl hydroxylase by fibroblasts, has been proposed as a drug for treating tissue disorders associated with fibroblast hyperproliferation and collagen accumulation (as is the case in fibrosis) [S. Murad and Pinnell, 1987, J. Biol. Chem., 262: 11973-11978; S. Murad et al., 1987, Arch. Biochem. Biophys., 292: 234-238; J. T. Handa et al., 1994, Invest. Ophthalmol. Vis. Sci., 35: 463-469; J. T. Handa et al., 1993, Invest. Ophthalmol. Vis. Sci., 34: 567-575; S. Murad et al., 1994, Arch. Biochem. Biophys., 308: 42-47]. With respect to the suppression of lysyl hydroxylase, it was suggested that xe2x80x9cThe rationale for this therapeutic approach to fibrosis is based on the consideration that a collagen deficient in hydroxylysine and therefore hydroxylysine-derived crosslinks would be deposited in the extracellular matrix as a nonfunctional protein with increased susceptibility to degradation by collagenase, thus limiting its amount in the fibrotic tissuexe2x80x9d [S. Murad et al., 1994, Arch. Biochem. Biophys., 308: 42-47]. In the context of this and other papers of the same group it is clear that the authors believe that lowering the hydroxylysine levels in the telopeptides results in decreased cross-link levels. The authors specifically stated that minoxidil reduces cross-link levels [J. T. Handa et al., 1994, Invest. Ophthalmol. Vis. Sci., 35: 463-469; J. T. Handa et al., 1993, Invest. Ophthalmol. Vis. Sci., 34: 567-575; S. Murad et al., 1994, Arch. Biochem. Biophys., 308: 42-47]xe2x80x94despite the lack of any experimental evidence for this statement. Such a statement is not necessarily true and as a matter of fact even unlikely. Firstly, hydroxyallysine-derived cross-links are likely to be replaced by allysine-derived cross-links. Secondly, although minoxidil inhibits mRNA levels of PLOD1 (thereby reducing Hyl levels of the triple helical part of collagen) [e.g. T. Hautala et al., 1992, Biochem. J., 283: 51-54], it has never been shown that minoxidil reduces the lysyl hydroxylation level in the telopeptides: it was at that time believed that lysyl hydroxylase encoded by PLOD1 was capable of hydroxylating the lysine residues in both the triple helix and the telopeptides of collagen molecules, which is not the case. The abbreviation PLOD is derived from procollagen-lysine, 2-oxoglutarate 5-dioxygenase (which is the systematic name of lysyl hydroxylase), the 1 indicates that it is the first discovered PLOD gene.
PLOD1 is the gene that is mutated in Ehlers-Danlos type VI syndrome (EDS-VI) [J. Brickmann et aL, 1998, Arch. Dermatol. Res., 290: 181-186], a disease that is biochemically characterized by a hydroxylysine deficiency of collagen. Close examination of EDS-VI patients revealed that, although collagen type I and III in most tissues are Hyl deficient, collagen type II and V are not [A. Ihme et al., 1984, J. Invest. Dermat., 83: 161-165]. Thus it appears, that collagen-type specific lysyl hydroxylases exist. The presence of such lysyl hydroxylases was also postulated by Risteli et al. [1980, Biochem. Biophys. Res. Commun., 96: 1778-1784], who found that lysyl hydroxylase of normal fibroblasts preferentially hydroxylated collagen type I, whereas the residual lysyl hydroxylase activity of EDS-VI fibroblasts was preferentially directed towards type IV collagen. In addition, EDS-VI patients show differences in the hydroxylation of collagen type I derived from various tissues: collagen type I from skin and bone are Hyl deficient, whereas collagen type I from tendon, kidney and lung was not [A. Ihme et al., 1984, J. Invest. Dermatol., 83: 161-165]. This indicates the existence of tissue-specific forms of collagen-type specific lysyl hydroxylases.
Recently, two other lysyl hydroxylases have been cloned, PLOD2 (SEQ ID NO. 7-12) and PLOD3 (SEQ ID NO. 13-16), with an overall amino acid sequence identity of 75% and 59% with that of PLOD1 (SEQ ID NO. 1-6), respectively. The three lysyl hydroxylases show a tissue-specific distribution [M. Valtavaara et al., 1997, J. Biol. Chem., 272: 6831-6834; M. Valtavaara et al., 1998, J. Biol. Chem., 273: 12881-12886; K. Passoja et al., 1998, Proc. Natl. Acad. Sci. USA, 95: 10482-10486]. PLOD2 also shows a tissue-specific splice variant [H. N. Yeowell and L. C. Walker, 1999, Matrix Biol., 18: 179-187]. Furthermore, there is some evidence at the DNA level that tissue-specific forms of PLOD1 exist [H. N. Yeowell et aL, 1994, J. Invest. Dermatol., 102: 382-384]. PLOD1-3 have been expressed in a baculovirus expression system; the proteins encoded by the cDNA exhibit activity towards the synthetic peptide containing the helical sequence IKGIKGIKG. Although the specificity of PLOD1-3 towards the different collagen types has so far not been investigated, the relatively low sequence homology indicates differences in the substrate properties of these enzymes.
Interestingly, EDS-VI patients show a normal level of pyridinolines in tissues (e.g. in collagen type I from bone) or a normal excretion level of pyridinolines in urine [B. Steinmann et al, 1995, Am. J. Hum. Genet., 57: 1505-1508]. Pyridinolines are cross-links derived from the hydroxyallysine route (FIG. 2). Thus, despite the deficiency of Hyl in the triple helix, a normal amount of Hyl is present in the telopeptides. This is circumstantial evidence for yet another set of lysyl hydroxylases, for which the term xe2x80x9ctelopeptide lysyl hydroxylasexe2x80x9d has been coined (as opposed to xe2x80x9chelical lysyl hydroxylasexe2x80x9d).
If a telopeptide lysyl hydroxylase indeed exists, one would expect that diseases exist that are characterized by an upregulation or downregulation of telopeptide lysyl hydroxylase, resulting in an increase or decrease of telopeptide hydroxylysine and, consequently, in an increase or decrease of hydroxyallysine-derived cross-links. It is shown herein that the underlying molecular defect of the Bruck syndrome (a heritable connective tissue disease) is the virtual absence of telopeptide hydroxylysine of collagen type I in bone, thus providing for the first time genetic evidence for the presence of telopeptide lysyl hydroxylase. Furthermore, in Bruck syndrome a normal level of telopeptide hydroxylysine of collagen type I in joint ligament is seen, thus providing for the first time genetic evidence that tissue-specific telopeptide lysyl hydroxylases exist. Moreover, it is shown herein that there is a telopeptide lysyl hydroxylase which is located on chromosome 17p12, and that PLOD2 (located on chromosome 3) encodes for a telopeptide lysyl hydroxylase (SEQ ID NO. 7-12). Furthermore, it is shown herein that collagen molecules cross-linked by means of hydroxyallysine cross-links have a different intrafibrillar packing than molecules cross-linked by means of allysine, that telopeptide lysyl hydroxylase is upregulated in scars and other fibrotic tissues and that collagen molecules crosslinked by hydroxyallysine are more resistant toward proteolytic enzymes, explaining at least in part the irreversibility of collagen accumulation in fibrosis. It is an object of the present invention to provide a new method for the prevention of excessive collagen accumulation in wound healing and in other processes in which fibrosis occurs as the final outcome by inhibiting the formation of hydroxyallysine-derived cross-links.
This invention provides a method of treating a fibrotic condition in a mammal suffering from said condition comprising administration to said mammal of an effective amount of a composition that selectively inhibits the activity or production of telopeptide lysyl hydroxylase.
The words xe2x80x9cselectively inhibits the activity or production of telopeptide lysyl hydroxylasexe2x80x9d are used herein in a broad sense, in that they not only cover the actual inhibition of the enzyme as such, but also cover a selective inhibition of the transcription of a telopeptide lysyl hydroxylase gene, selective inhibition of the translation of mRNA derived from a telopeptide lysyl hydroxylase gene, and treatment with (a recombinant gene coding for) a mutated telopeptide lysyl hydroxylase that shows no activity towards telopeptides but that is competitive to endogenous telopeptide lysyl hydroxylase with respect to its natural substrate (collagen telopeptides).
The method of the invention is broadly applicable with mammals of any kind, but in a preferred embodiment said mammal is a human being.
Other objects, features and advantages of the present invention will become apparent as the description proceeds.